Project Summary The Eph family of receptor tyrosine kinases, the largest in the mammalian genome, and their ephrin ligands critically influence inflammation in a variety of pathological conditions. Our work provided the first description of EphA2 expression in multiple cell types in the atherosclerotic plaque, and we recently demonstrated that EphA2 deletion reduces atherosclerotic plaque formation associated with diminished plaque inflammation. Surprisingly, EphA2 deletion also reduced progression to advanced atherosclerotic disease with diminished plaque smooth muscle content. Phenotypic modulation of smooth muscle cells from a contractile to a synthetic phenotype drives their accumulation during plaque development, and smooth muscle cells show enhanced EphA2 expression during phenotypic modulation both in vitro and in vivo. Our preliminary data show that matrix remodeling that promotes fibronectin-dependent integrin signaling critically regulates EphA2 expression during phenotypic modulation, whereas blunting EphA2 expression limits fibronectin matrix deposition in vivo and in vitro. Taken together, these data suggest a dynamic interplay between EphA2 and matrix remodeling that critically regulates the smooth muscle matrix deposition. In addition to matrix deposition, we also demonstrated a critical role for EphA2 expression in smooth muscle mitogenic signaling (ERK1/2, AKT) and proliferation both in vitro and in atherosclerotic plaques in vivo. While EphA2 ligation by ephrinA1 elicits multiple aspects of EphA2's atherogenic proinflammatory responses, our preliminary data show that EphA2 ligation reduces smooth muscle proliferation. Conversely, EphA2 can also signal in a ligand-independent state, which promotes cell proliferation and migration in cancer models, and we observed an increase in EphA2 ligand-independent signaling (Ser897 phosphorylation) during smooth muscle proliferation in vitro and at sites of smooth muscle phenotypic modulation in vivo. However, the mechanisms regulating EphA2's differential ligand-dependent and ligand-independent mitogenic signaling remain virtually unexplored. Therefore, we hypothesize that dynamic interplay between EphA2 expression and matrix remodeling in the atherosclerotic plaque drives smooth muscle invasion and fibroproliferative remodeling through activation of EphA2 ligand-independent signaling. To test this hypothesis, we will characterize the dynamic interplay between EphA2 expression and matrix composition using both cell culture models and smooth muscle- specific deletion of fibronectin and fibronectin-binding integrins (Aim 1), we will determine the mechanisms by which EphA2 signaling affects smooth muscle phenotype (Aim 2), and we will assess the role of EphA2 cell-type specific expression and signaling in atherosclerotic fibroproliferative remodeling using novel EphA2 conditional knockouts and inhibitors of EphA2 ligation and kinase activity. Successful completion of these Aims will identify a novel mechanism to selectively reduce plaque-associated inflammation while promoting smooth muscle- dependent stabilization of the fibrous cap.